OBJECTIVE. To investigate a nosocomial outbreak of infection with Acinetobacter strain RUH 1139, in the unit of high neonatal risk at University Hospital of The Andes (Mérida, Venezuela). METHODS. Twenty-eight Acinetobacter strains were detected by biochemical testing and further identified to the species level by examination of the gene encoding 16S ribosomal DNA, using restriction analysis and gene sequencing. The epidemiological relationship between the strains was established by means of repetitive extragenic palindromic polymerase chain reaction (REP-PCR) and pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibilities were determined by disk diffusion. RESULTS. The spread of an epidemic strain of Acinetobacter RUH 1139 among 16 patients over a period of 3 months was demonstrated using antimicrobial susceptibility testing, PFGE, and REP-PCR. The epidemic strain was also isolated in 2 of the sampled parenteral nutrition solutions. All the patients involved in the infection outbreak had received parenteral solution. Moreover, strains of Acinetobacter RUH 1139 with another PFGE pattern and of A. baumannii were sporadically isolated before and during the outbreak. CONCLUSION. This is the first description of an outbreak of infection with this genospecies of Acinetobacter in which parenteral nutrition solution was potentially the infection source. © 2006 by The Society for Healthcare Epidemiology of America. All rights reserved.